J. HARRIETT MCCOY- AND MARY ALICE Food and Nutrition Department, Iowa State University, Ames, Iowa 50010
KENNEY "
ABSTRACT The effects of dietary magnesium on growth, food efficiency, organ development, splenic nucleic acids, and serum antibody were studied in two experi ments with male Wistar rats. Diets containing 30% protein from casein were fed ad libitum. Rats were immunized intravenously with sheep red blood cells. Blood was obtained 5 and 9 days after immunization. In experiment 1, a group of weanling rats was fed 10 ppm Mg for 8 days, followed by 142 ppm for 37 days. Group 2 (controls) was fed 480 ppm Mg for 45 clays. Group 1 weighed less but had larger spleens, kidneys, and testes relative to body size than did group 2. Nucleic acids per gram spleen were similar in both groups as were serum 7-globulin and its 19S and 7S components. Anti body log titers for group 1 were 45 and 65% of control agglutinin levels and 44 and 80% of control hemolysin values on days 5 and 9, respectively. In experiment 2, 200-g rats were fed 10 ( group 3 ) or 480 ppm ( group 4 ) Mg for 38 days. Most effects of the 10 ppm Mg diet were similar to those seen in magnesium deficiency in experiment 1. Antibody titers for group 3 were 30 and 25% of control agglutinin and 43 and 53% of control hemolysin values on days 5 and 9, respectively. Total serum 7-globulin and its 19S fraction were similar in both groups, while the 7S fraction of group 3 was only 64% of the control value. J. Nutr. 105: 791-797, 1975. INDEXING 7-globulin
KEY WORDS
antibody
magnesium
Numerous studies have demonstrated roles for dietary protein and lipid as well as for singular amino acids and vitamins in the immune responses of experimental animals. Assessment of the effect of min eral intake on the immune response has been limited4 (1-4). Because malnutrition in man is likely to involve multiple nutrient deficiencies, it seems important to investi gate roles for mineral elements in resist ance mechanisms. Synthesis of antibodies, major components of the total resistance system, is regulated by the supply and uti lization of factors involved in protein syn thesis. Magnesium, a major intracellular cation, has been linked experimentally with protein metabolism in several ways. It reportedly activates enzymes critical to the formation of ATP needed for peptide bond synthesis (5). It is required for acti vation and transfer of amino acids to solu ble RNA (6) and for stabilization of DNA,
immune
response
RNA, and ribosomes (7, 8). In addition, magnesium reportedly affects tissue growth in the thymus (9), which is involved with the establishment of immunologically com petent cells. The relation of magnesium to protein synthesis as a part of resistance mecha nisms has received little attention. Alcock and Shils (4) reported decreased levels of immunoglobulin G (IgG or 7S antibody) in serum or plasma of magnesium-depleted rats. When these animals were repleted, marked increases in serum IgG occurred immediately. Because it is the major comReeelved for publication December 26, 1074. 'Journal Tuner no. .1-8069. Iowa Agriculture and Home Economics Experiment Station. Amen, Iowa Project no. 1717. 2 Present address: Department of Ilonir Economics. University of Arkansas. Fayetteville. Ark. 72701. 'Prêtent address: Department of Food and Nutri tion. Texas Tech I'niversity. Luhhock, Tex. 7!»4OJ| 1 Hitchincs, f}. H.. Falco. E. A. & Sherwood. M. B. (194!)) Dietary manganese and susceptibility to pneumococeal infection. Federation I'roc. 8, 1MI7. (Abstr.) 791
Downloaded from https://academic.oup.com/jn/article-abstract/105/6/791/4768848 by University of California, San Francisco user on 01 January 2019
Depressed Immune Response in the Magnesium-deficient Rat1
792
J. HARRIETT
MCCOY AND MARY ALICE KENNEY
PROCEDURES
Our purpose was to compare the im mune responses of rats fed low magnesium diets with those of control rats. In both experiments, male rats of the Wistar strain5 were fed a basal diet containing (in %)-. D-glucose, 57.1; vitamin test ca sein, 32.9; corn oil, 5.0; salt mix (reagentgrade chemicals), 2.8; S-T nonnutritive fiber, 2.0; DL-methionine, 0.2. Each rat re ceived daily 1 ml of vitamin mixture8 in 20% ethanol plus 1.0 mg of DL-o-tocopherol in 50 mg corn oil and 50 mg of cod-liver oil9 apart from the diet. Diets and deionized water were provided ad libitum in both experiments. Rats were housed individually in stainless-steel, wire-mesh hanging cages on racks in a temperatureand humidity-controlled room. They were weighed 2 to 3 times a week, and weekly food intake records were kept. Rats were anesthetized with ether and were im munized with 1 ml of a 2% suspension of sheep red blood cells 10 (SRBC) in 0.85% saline injected through a lateral caudal vein with a 25-gauge sterile needle. They were bled from a tail incision prior to autopsy at times designated under the specific experiments. At autopsy, rats were killed with an intraperitoneal injection of sodium pentobarbital solution. Blood was withdrawn from the abdominal aorta, and the serum was separated and frozen for analysis of specific antibody and -/-globulin fractions at a later time. Agglutinin and hemolysin activities of the immune sera were titrated using a semi-micro method described by Kenney et al. (10). The end point for agglutinin activity was recorded as the highest dilution of serum showing positive agglutination of SRBC. The end point for hemolysin activity was recorded as the dilution at which half of the SRBC were lysed. Results for both antibodies were expressed as the logarithms of the reciprocals of these dilutions. A modifica tion of the method described bv Friedman (11) was used for extraction of y-globulin
from 0.3 ml of serum. The globulin was precipitated with a solution 1.4 M (33% saturated) in ammonium sulfate and 0.5 M in sodium chloride at pH 6.4 in the cold, and the supernatant was removed after centrifugation. The precipitate was dis solved and phosphate made to abuffer, volumepHof7.8 1 ml Sorensen's (12).in Column chromatography at room tempera ture effected separation of fractions corre sponding to 19S and 7S antibodies " from the total y-globulin solution. A 30 X 1.5 cm column with the corresponding eluent reservoir was packed with Sepharose-6B;la phosphate buffer (12) was used as the eluent. For antibody separation, 0.5 ml of the y-globulin solution was added to the column. The sample was eluted at a flow rate of three drops per minute. Thirtyeight fractions of 1.5 ml (50 drops) were collected for each sample. The protein content of each fraction was estimated by recording the ultraviolet absorption at 280 nm and comparing it with a standard curve prepared from readings for standard y-globulin13 solutions. Diets were ashed in a muffle furnace and were analyzed for magnesium by atomic absorption spectrophotometry at 284 nm. Analysis of vari ance and F tests were used to evaluate differences between group means. Differ ences were considered to be significant when P < 0.05 unless indicated otherwise. Experiment 1 compared the immune re sponses of 2 groups of 10 male weanling rats. Group 1 was fed a very low mag nesium diet (10 ppm Mg) for 8 days postweaning followed by a low magnesium 6 Iowa State University stock colony. Original breeding stock obtained from Slmonsen Laboratories, White Bear Lake, Minn. « Salt content of diet (g/kg) : CaHPO,. 20.3fiS7 ; CuSO,, 0.0126; MgSO,, 1.9798; KI, 0.0003; K¡,HPO4. 4.MIÜI4;Nad, 1.2711; ZnSO,-7H2O, 0.0528; FeSO47H..O. 0.1244; MnSO.-HsO, 0.1538. 7 MgSOt was omitted or reduced In diets Inade quate In magnesium. 8 Calculated to provide dug/day) : thlamln HC1, 20.0; rlboflavin, 39.0; pyridoxine HC1, 20.0; folle acid, 20.0 ; calcium pantothenate, 97.0 ; p-amlnobenzolc acid, 97.0; vitamin B-12, 0.2; blotln, 2.0; niactn, 200.0 ; (mg/day) : inosltol, 2.4 ; chollne chloride, 4.8. 9 50 mg cod-liver oil provided 42.5 and 4.25 Ü.S.P. units of vitamins A ad D, respectively. 10Baltimore Biological Laboratories, Westchester, Penn. 11Wong, M. P. (1971) Immune response and y-globulin metabolism of rats fed different sulfurcontaining amino acids. M.S. Thesis, Iowa State Uni versity, Ames, Iowa. u Sephadex with 6% agarose added ; Pharmacia Fine Chemicals. Piscataway, N.J. "Rat 7-globulln, fraction II, Pentex, Inc.. Kankakee, 111.
Downloaded from https://academic.oup.com/jn/article-abstract/105/6/791/4768848 by University of California, San Francisco user on 01 January 2019
ponent of y-globulin, IgG concentration gives an index to total antibody formation. Two experiments in our laboratory assessed the effects of dietary magnesium deficiency on production of specific antibody in mag nesium-depleted rats.
793
IMMUNE RESPONSE IN MAGNESIUM DEFICIENCY TABLE 1 Body and organ weights Group
Body wt
Spleen
Kidney
Liver
Testes
weight1.70±0.132 body
122
4.00±0.11
±5230
0.25±0.010.48±0.032 0.88±0.02 4.04±0.101.27±0.072
±5« 34a247 348±40.39±0.032 0.21±0.01% 1 Mean ±SEM.
1.33 ±0.051.63±0.0(i2
0.85±0.023.75±0.12 3.68±0.071.88±0.082 0.97±0.04
2 Different from control group in the same experiment, P
KENNEY "
ABSTRACT The effects of dietary magnesium on growth, food efficiency, organ development, splenic nucleic acids, and serum antibody were studied in two experi ments with male Wistar rats. Diets containing 30% protein from casein were fed ad libitum. Rats were immunized intravenously with sheep red blood cells. Blood was obtained 5 and 9 days after immunization. In experiment 1, a group of weanling rats was fed 10 ppm Mg for 8 days, followed by 142 ppm for 37 days. Group 2 (controls) was fed 480 ppm Mg for 45 clays. Group 1 weighed less but had larger spleens, kidneys, and testes relative to body size than did group 2. Nucleic acids per gram spleen were similar in both groups as were serum 7-globulin and its 19S and 7S components. Anti body log titers for group 1 were 45 and 65% of control agglutinin levels and 44 and 80% of control hemolysin values on days 5 and 9, respectively. In experiment 2, 200-g rats were fed 10 ( group 3 ) or 480 ppm ( group 4 ) Mg for 38 days. Most effects of the 10 ppm Mg diet were similar to those seen in magnesium deficiency in experiment 1. Antibody titers for group 3 were 30 and 25% of control agglutinin and 43 and 53% of control hemolysin values on days 5 and 9, respectively. Total serum 7-globulin and its 19S fraction were similar in both groups, while the 7S fraction of group 3 was only 64% of the control value. J. Nutr. 105: 791-797, 1975. INDEXING 7-globulin
KEY WORDS
antibody
magnesium
Numerous studies have demonstrated roles for dietary protein and lipid as well as for singular amino acids and vitamins in the immune responses of experimental animals. Assessment of the effect of min eral intake on the immune response has been limited4 (1-4). Because malnutrition in man is likely to involve multiple nutrient deficiencies, it seems important to investi gate roles for mineral elements in resist ance mechanisms. Synthesis of antibodies, major components of the total resistance system, is regulated by the supply and uti lization of factors involved in protein syn thesis. Magnesium, a major intracellular cation, has been linked experimentally with protein metabolism in several ways. It reportedly activates enzymes critical to the formation of ATP needed for peptide bond synthesis (5). It is required for acti vation and transfer of amino acids to solu ble RNA (6) and for stabilization of DNA,
immune
response
RNA, and ribosomes (7, 8). In addition, magnesium reportedly affects tissue growth in the thymus (9), which is involved with the establishment of immunologically com petent cells. The relation of magnesium to protein synthesis as a part of resistance mecha nisms has received little attention. Alcock and Shils (4) reported decreased levels of immunoglobulin G (IgG or 7S antibody) in serum or plasma of magnesium-depleted rats. When these animals were repleted, marked increases in serum IgG occurred immediately. Because it is the major comReeelved for publication December 26, 1074. 'Journal Tuner no. .1-8069. Iowa Agriculture and Home Economics Experiment Station. Amen, Iowa Project no. 1717. 2 Present address: Department of Ilonir Economics. University of Arkansas. Fayetteville. Ark. 72701. 'Prêtent address: Department of Food and Nutri tion. Texas Tech I'niversity. Luhhock, Tex. 7!»4OJ| 1 Hitchincs, f}. H.. Falco. E. A. & Sherwood. M. B. (194!)) Dietary manganese and susceptibility to pneumococeal infection. Federation I'roc. 8, 1MI7. (Abstr.) 791
Downloaded from https://academic.oup.com/jn/article-abstract/105/6/791/4768848 by University of California, San Francisco user on 01 January 2019
Depressed Immune Response in the Magnesium-deficient Rat1
792
J. HARRIETT
MCCOY AND MARY ALICE KENNEY
PROCEDURES
Our purpose was to compare the im mune responses of rats fed low magnesium diets with those of control rats. In both experiments, male rats of the Wistar strain5 were fed a basal diet containing (in %)-. D-glucose, 57.1; vitamin test ca sein, 32.9; corn oil, 5.0; salt mix (reagentgrade chemicals), 2.8; S-T nonnutritive fiber, 2.0; DL-methionine, 0.2. Each rat re ceived daily 1 ml of vitamin mixture8 in 20% ethanol plus 1.0 mg of DL-o-tocopherol in 50 mg corn oil and 50 mg of cod-liver oil9 apart from the diet. Diets and deionized water were provided ad libitum in both experiments. Rats were housed individually in stainless-steel, wire-mesh hanging cages on racks in a temperatureand humidity-controlled room. They were weighed 2 to 3 times a week, and weekly food intake records were kept. Rats were anesthetized with ether and were im munized with 1 ml of a 2% suspension of sheep red blood cells 10 (SRBC) in 0.85% saline injected through a lateral caudal vein with a 25-gauge sterile needle. They were bled from a tail incision prior to autopsy at times designated under the specific experiments. At autopsy, rats were killed with an intraperitoneal injection of sodium pentobarbital solution. Blood was withdrawn from the abdominal aorta, and the serum was separated and frozen for analysis of specific antibody and -/-globulin fractions at a later time. Agglutinin and hemolysin activities of the immune sera were titrated using a semi-micro method described by Kenney et al. (10). The end point for agglutinin activity was recorded as the highest dilution of serum showing positive agglutination of SRBC. The end point for hemolysin activity was recorded as the dilution at which half of the SRBC were lysed. Results for both antibodies were expressed as the logarithms of the reciprocals of these dilutions. A modifica tion of the method described bv Friedman (11) was used for extraction of y-globulin
from 0.3 ml of serum. The globulin was precipitated with a solution 1.4 M (33% saturated) in ammonium sulfate and 0.5 M in sodium chloride at pH 6.4 in the cold, and the supernatant was removed after centrifugation. The precipitate was dis solved and phosphate made to abuffer, volumepHof7.8 1 ml Sorensen's (12).in Column chromatography at room tempera ture effected separation of fractions corre sponding to 19S and 7S antibodies " from the total y-globulin solution. A 30 X 1.5 cm column with the corresponding eluent reservoir was packed with Sepharose-6B;la phosphate buffer (12) was used as the eluent. For antibody separation, 0.5 ml of the y-globulin solution was added to the column. The sample was eluted at a flow rate of three drops per minute. Thirtyeight fractions of 1.5 ml (50 drops) were collected for each sample. The protein content of each fraction was estimated by recording the ultraviolet absorption at 280 nm and comparing it with a standard curve prepared from readings for standard y-globulin13 solutions. Diets were ashed in a muffle furnace and were analyzed for magnesium by atomic absorption spectrophotometry at 284 nm. Analysis of vari ance and F tests were used to evaluate differences between group means. Differ ences were considered to be significant when P < 0.05 unless indicated otherwise. Experiment 1 compared the immune re sponses of 2 groups of 10 male weanling rats. Group 1 was fed a very low mag nesium diet (10 ppm Mg) for 8 days postweaning followed by a low magnesium 6 Iowa State University stock colony. Original breeding stock obtained from Slmonsen Laboratories, White Bear Lake, Minn. « Salt content of diet (g/kg) : CaHPO,. 20.3fiS7 ; CuSO,, 0.0126; MgSO,, 1.9798; KI, 0.0003; K¡,HPO4. 4.MIÜI4;Nad, 1.2711; ZnSO,-7H2O, 0.0528; FeSO47H..O. 0.1244; MnSO.-HsO, 0.1538. 7 MgSOt was omitted or reduced In diets Inade quate In magnesium. 8 Calculated to provide dug/day) : thlamln HC1, 20.0; rlboflavin, 39.0; pyridoxine HC1, 20.0; folle acid, 20.0 ; calcium pantothenate, 97.0 ; p-amlnobenzolc acid, 97.0; vitamin B-12, 0.2; blotln, 2.0; niactn, 200.0 ; (mg/day) : inosltol, 2.4 ; chollne chloride, 4.8. 9 50 mg cod-liver oil provided 42.5 and 4.25 Ü.S.P. units of vitamins A ad D, respectively. 10Baltimore Biological Laboratories, Westchester, Penn. 11Wong, M. P. (1971) Immune response and y-globulin metabolism of rats fed different sulfurcontaining amino acids. M.S. Thesis, Iowa State Uni versity, Ames, Iowa. u Sephadex with 6% agarose added ; Pharmacia Fine Chemicals. Piscataway, N.J. "Rat 7-globulln, fraction II, Pentex, Inc.. Kankakee, 111.
Downloaded from https://academic.oup.com/jn/article-abstract/105/6/791/4768848 by University of California, San Francisco user on 01 January 2019
ponent of y-globulin, IgG concentration gives an index to total antibody formation. Two experiments in our laboratory assessed the effects of dietary magnesium deficiency on production of specific antibody in mag nesium-depleted rats.
793
IMMUNE RESPONSE IN MAGNESIUM DEFICIENCY TABLE 1 Body and organ weights Group
Body wt
Spleen
Kidney
Liver
Testes
weight1.70±0.132 body
122
4.00±0.11
±5230
0.25±0.010.48±0.032 0.88±0.02 4.04±0.101.27±0.072
±5« 34a247 348±40.39±0.032 0.21±0.01% 1 Mean ±SEM.
1.33 ±0.051.63±0.0(i2
0.85±0.023.75±0.12 3.68±0.071.88±0.082 0.97±0.04
2 Different from control group in the same experiment, P
